The present invention relates to the use of antisense cDNA targeting for the inhibition and/or prevention of tumor growth. More specifically, this invention relates to the use of antisense nucleic acid derived from the antisense cDNA sequence of tubedown-1 for the inhibition and/or prevention of bone tumors, especially osteosarcoma and Ewings Sarcoma family of tumors.
Cancer is generally treated with cytoreductive therapies that involve administration of ionizing radiation or chemical toxins that kill rapidly dividing cells. Unfortunately, these therapies are highly toxic to non-cancer cells and cause severe side effects, such as bone marrow suppression, hair loss and gastrointestinal disturbances.
Osteosarcoma, a bone cancer occurring primarily in teenagers and young adults, affects approximately 2100 individuals yearly in the United States (1). This malignancy accounts for as many as 5% of all childhood malignancies and 60% of all malignant childhood bone tumors (2). Despite radical surgical resection of the primary tumor and aggressive adjuvant chemotherapy, the overall 2-year metastasis-free survival rate approaches only 66%. More than 30% of patients with this disease develop lung metastasis within the first year (3-4). The survival rate among those affected with osteosarcoma has not changed significantly over the past 10 years, despite changes in adjuvant chemotherapy (5).
Ewing""s Sarcoma is the second most frequent type of bone tumor and Ewing""s Sarcoma most often strikes during the second decade of life. Tumors can metastasize to lungs, other bones and to the bone marrow (6-8). Approximately 25% of Ewing""s tumor patients present detectable metastatic disease at diagnosis, but it is probable that most patients have micrometastases (9-10). Approximately 40 percent of Ewing""s sarcoma patients do not survive. Extraosseous Ewing""s sarcoma variants including the peripheral primitive neuroectodermal tumor (PNET) and the Askin tumor (a thoracic form of Ewing""s sarcoma) are more rare (7-8).
Chemotherapy using cytotoxic drugs (vincristine, actinomycin-D, cyclophosphamide, etoposide), followed by surgery or radiotherapy is the current treatment regimen for Ewing""s sarcomas (6, 10). There is a 50-70% survival rate (5 years) in cases of localized disease. However, when metastatic disease is observed, there is a 19-30% survival rate (5 years) (10). Moreover, the risk of developing secondary malignancies is approximately 6.7% at 10 years and 43% at 20 years, and relapse 5 years after treatment occurs in approximately 9-16% of cases (7, 9). The intensive radiation and chemotherapy treatments for both osteosarcoma and Ewing""s sarcoma tumors are associated with a high degree of toxicity. Thus, new treatments are needed for these types of pediatric bone tumors.
Although the genetic alterations involved in Ewing""s sarcoma have been identified, understanding how these pathologically modified genetic pathways lead to and support growth of Ewing""s tumors have not been completely realized. The isolation, characterization and practical manipulation of additional regulatory molecules which could play a common role in the growth control of Ewing""s sarcoma cells may lead to new and improved therapies for the Ewing""s family of tumors. Such new therapy would be aimed at altering the signaling pathways which regulate growth of Ewing""s tumor cells and could offer alternatives or supplements to the currently available treatments.
Furthermore, even less is known about the genetics or growth control of osteosarcomas. The isolation, characterization and practical manipulation of master regulatory molecules which could play a common role in the growth control of bone tumor cells may lead to new and more effective therapies for bone cancer. New treatments aimed at altering the signaling pathways which regulate growth of bone tumor cells offers alternatives or supplements to the exclusive use of radiotherapy, cytotoxic chemotherapeutic drugs and surgery.
The inventors have cloned a new gene named tubedown-1 (tbdn-1) which encodes a novel protein associated with an acetyltransferase activity (11). This tubedown-1 protein is highly expressed in primitive bone tumors of mesenchymal and neuroectodermal origin such as osteosarcoma and Ewing""s sarcoma. The in vivo expression pattern of tbdn-1 suggests it may play a role in regulating endothelial, hematopoietic and bone development (FIG. 1) (11). In early myeloid blood cells, blood vessel endothelium and bone, tbdn-1 is expressed at high levels early on and becomes downregulated as these cells mature (11). Tbdn-1 expression distribution during embryogenesis is similar to the expression distribution of FLI-1 (12). In adults, tbdn-1 is restricted to ocular and ovarian blood vessels, bone marrow and atrial endocardium.
Tbdn-1 is also expressed highly in Ewing""s sarcoma cells, suggesting that it may play a regulatory role in this type of tumor. This finding implies that tbdn-1 may play a regulatory role in these types of bone cancer and perhaps bone cancer in general. Therefore, one approach to the treatment of these sarcomas would be a gene therapy approach aimed to block expression of tbdn-1 in these bone tumors by inducing expression of an antisense tbdn-1 cDNA fragment (AStbdn-1) which inhibits tumor growth.
As described herein, the present invention comprises a method to use tbdn-1 antisense reagents as gene therapy agents for the treatment of bone tumors and Ewing""s sarcoma family of tumors. Antisense-based reagents, such as tbdn-1 antisense construct or biologically stabilized oligonucleotides, or any compound which would elicit the downregulation of tbdn-1 level or activity and the same biological effects as tbdn-1 antisense construct on bone tumor growth in vivo provide valuable alternative or supplemental therapies for bone cancer.
The antisense cDNA molecules utilized in the present invention generate antisense mRNA of at least 70% complementarity to mRNA produced by a native tubedown-1 gene. Preferred antisense oligonucleotide molecules are selected from the group consisting of SEQ ID NO. 3 and SEQ ID NO. 4. These above-cited antisense oligonucleotides derived from the cDNA sequence of SEQ ID NO. 2, may also be formulated as compositions comprising a safe and effective amount of an antisense oligonucleotide molecule and a pharmaceutically acceptable carrier.
A gene therapy approach for treatment of mammals afflicted with bone tumors, such as Ewing""s Sarcoma and osteosarcoma or expressing a tubedown-1 protein is provided. For this method of treatment, a biologically active antisense cDNA is generated from the cDNA of SEQ ID NO. 2 and administered to cells of an individual producing excess of a tubedown-1 gene.
This method further comprises in vivo administration into host cells a replicable vector comprising and expressing the desired antisense cDNA, which in turn produces the antisense mRNA. The vector is then taken up by the cells to produce the antisense mRNA. This antisense mRNA binds to native mRNA produced by the tubedown-1 gene, thereby blocking expression of the gene. The antisense cDNA generates antisense mRNA of at least 70% complementarity to mRNA produced by a native tubedown-1 gene and which can hybridize with the native mRNA under low and high stringency conditions. The preferred antisense cDNA""s for use in this gene therapy treatment are selected from the group consisting of SEQ ID NO. 3 and SEQ ID NO. 4 and mixtures thereof.
In an alternative treatment for bone tumors, and Ewings Sarcoma family of tumors and osteosarcoma in particular, single-stranded antisense oligonucleotides derived from the antisense cDNA sequence of SEQ ID NO. 3 or 4 can be generated ex vivo. These antisense oligonucleotides are at least 15 nucleobases in length and preferably at least 25 nucleobases in length. Thus, when introduced into the cell, these antisense oligonucleotides cause inhibition of expression of tbdn-1 by hybridizing with native mRNA and genomic sequences of a tbdn-1 gene. Other biological or chemical factors to inhibit the expression of said tbdn-1 protein are also within the scope of this invention.